In virtually all professional and social spheres, impaired hearing and thus a restricted ability to communicate have considerable implications on the quality of life. The sensory disorder “hardness of hearing” is one of the most urgent health problems in a society depending on communication.
On average, hardness of hearing affects about 10% of the population of the industrialized nations. In Germany, there are estimated to be 16 million people who are hard of hearing, almost one fifth of the total population (ifo-Institut, 1986). Thus, hardness of hearing is not only the most frequent disorder of a sensory organ, but also one of the most frequent chronic disorders in general.
Looking at the causes of hardness of hearing, in about 80% of the cases, the people affected suffer from sensorineural hearing loss. One of the most frequent reasons for this is the loss of sensory hair cells in the organ of Corti, the auditory sensory epithelium which, due to exposure to noise, side-effects of ototoxic medicaments, age-related degeneration or genetic causes are lost irreversibly by cell death (Nadol, 1993).
Hitherto, only the prosthetic provision of hearing aids can be offered for this most frequent form of hearing loss. However, for the persons affected, the result of this provision is often unsatisfactory due to the lack of speech recognition. Accordingly, hearing aids are actually used by only a relatively small proportion of the hard of hearing.
To date, there is no curative medical treatment option for the main cause of sensorineural hearing loss. Such a causal treatment would be possible only by replacing or regenerating the lost sensory hair cells of the organ of Corti.
In most mammalian organs and tissues, the ability for regeneration after damage is limited, or not present at all. Only very few organs and tissues such as, for example, liver, bones or skin have, over the entire lifetime of the organism, the ability for spontaneous regeneration by forming new cells. In many cases, the corresponding cells in the highly specialized organs and tissues (for example, heart, brain, skeletal muscle or the sensory epithelia of eye and inner ear) leave the cell cycle irreversibly to remain in a terminally differentiated state. As a consequence, these tissues also lose their ability to spontaneously regenerate in the case of damaging events. Accordingly, this leads to irreversible functional deficits. Thus, for example, a myocardial infarct in the case of the heart or a stroke in the case of the brain often means irreversible damage of the tissue areas affected, with corresponding permanent loss of function.
There are, however, for example, in amphibians, tissues and organs, exemplified by retina and extremities, where there is terminal differentiation, but which are nevertheless capable of spontaneous in vivo regeneration (Tsonis, 2000; Tsonis, 2002). The central cell biological event in these examples is cellular dedifferentiation, which allows generation of multipotent precursor cells from which regenerated cells may be formed by proliferation and redifferentiation.
Thus, dedifferentiation plays the decisive role in regeneration of terminally differentiated tissue of amphibious animals. In contrast, other vertebrates have low regeneration abilities.
Until about 20 years ago, for the hearing organ of mammals and birds, it was assumed that the sensory hair cells in the inner ear can be formed only during a short critical phase of embryonic development (Ruben, 1967). After this phase, the sensory epithelia were thought to be postmitotic and therefore not able to regenerate their sensory cells. However, surprisingly it was discovered that, after acoustic trauma and ototoxic damage, avian cochlea are capable of spontaneously regenerating sensory hair cells (Cotanche 1987; Cruz et al., 1987).
Cell division of supportive cells directly adjacent to the destroyed sensory hair cells has been described as the basic biological mechanism for sensory hair cell regeneration in avian cochlea (Corwin and Cotanche, 1988; Ryals and Rubel, 1988), where a population of undifferentiated cells is formed which are capable of redifferentiation to newly formed sensory hair cells and supportive cells. The result is the virtually complete morphological and functional recovery of the sensory epithelium in birds (Cotanche, 1999; Smolders, 1999).
Although this was initially obvious, due to fundamental cell biological differences, it has hitherto not been possible to apply findings from other models to mammals.
Corresponding experiments concerning regeneration of sensory hair cells in mammals gave no (Roberson and Rubel, 1994; Vago et al., 1998; Daudet et al., 1998; Daudet et al., 2002; Yamasoba et al., 2003) or very few (Yamasoba and Kondo, 2006) indications of a capability for spontaneous cell division of supportive cells in the organ of Corti. In particular, even after administration of growth factors, there are no indications of an inducible proliferation of supportive cells in the organ of Corti (Staecker et al., 1995; Daudet et al., 2002). Various experiments with cultures of early development stages of the organ of Corti of embryonal mice likewise showed, after defined laser damage, only individual proliferative events (Kelley et al., 1995).
This total lack of cell divisions suggests that the highly specialized supportive cell populations in the normal adult organ of Corti have reached a terminally differentiated state and are unable to re-enter the cell cycle. Thus, in the case of the inner ear, even a single acoustic trauma may result in the destruction of sensory hair cells, followed by unavoidable and irreversible loss of hearing.
It had recently been found that an extract can be obtained from amphibian tissues (for example, extremities) undergoing regeneration which is capable of inducing dedifferentiation even in mammalian cells (McGann et al., 2001). Using this extract, with appropriate stimulation, the dedifferentiation-based mechanism for regeneration of terminally differentiated cells can be transferred from amphibians to mammals (Odelberg, 2002). However, regeneration extracts mentioned are “protein cocktails” and details with regard to their composition are not known.
However, in the meantime, it has also been possible to achieve a corresponding effect in mammalian muscle cells using a defined low-molecular-weight compound (Chen et al., 2004). By screening, is was furthermore possible to identify several low-molecular-weight compounds producing regeneration biology-relevant effects in various cell types including glia cells (reviews in Xu et al., 2008; Schugar et al., 2008; Feng et al., 2009; Li and Ding, 2009). These effects suggest that it may also be possible to induce dedifferentiation-based regeneration in further cell types using suitable low-molecular-weight compounds (Tsonis, 2004; Kim et al., 2004; Odelberg, 2002).
Hitherto, the transfer of this concept to regeneration biological studies on the inner ear has been unique. Low-molecular-weight compounds capable of effecting sensory hair cell regeneration in the inner ear are neither known nor patented.
As yet, other concepts pursued in the current art for regenerating sensory hair cells in the organ of Corti likewise show little promise with regard to clinical application.
In the modulation of cell cycle regulation of supportive cells by switching off the cell cycle inhibitor p27Kip1, it was possible to achieve cell divisions in vivo (WO 99/42088). However, differentiation to sensory hair cells has hitherto only been observed under in vitro conditions outside of a tissue context (White et al., 2006).
In an in vivo model with induced sensory hair cell loss, gene therapeutically induced transdifferentiation of supportive cells with the transcription factor Math1, essential for sensory hair cell differentiation, led to conversion of supportive cells into sensory hair cells, even with partial functional recovery of the organ function (Izumikawa et al., 2005; Kawamoto et al., 2003). However, reduction in the number of supportive cells resulted in functional limitations for the organ of Corti as normal functioning of the complex micromechanic in the transduction process is impossible without supportive cells.
On activation of endogenous progenitor stem cells residing in the organ or of exogenous administration of heterologous stem cells to the inner ear, promising results were obtained (Tateya et al., 2003; Naito et al., 2004; Martinez-Monedero et al., 2007a, b; Li et al., 2003). However, a targeted or functionally relevant transplantation of stem cells into the inner ear has hitherto not been realized.
Accordingly, it could be helpful to identify low-molecular-weight compounds which stimulate an endogenous regeneration of terminally differentiated cells in highly specialized organs, tissues and sensory epithelia in mammals in situ. In particular, it could be helpful if these compounds could allow restoration of hearing in mammals by de novo formation of sensory hair cells in the adult organ of Corti. It could also be helpful for the first time to treat the causes of inner ear hardness of hearing on the basis of a pharmaceutical having regeneration biological activity.